Identification and characteristics of Pantoea cypripedii 4A strain producing high molecular exopolysaccharide

Searching for new microorganisms with great potential for the synthesis of high-molecular exopolysaccharides possessing some useful traits is very important in the scope of certain industries. The bacterial strain Pantoea cypripedii 4A isolated by direct plating on a rich agar medium was studied in this work as a potential producer of extracellular polysaccharides. This strain was isolated from forest litter mixed with the topsoil of the M.A. Zablotsky Prioksko-Terrasny Reserve. The Pantoea cypripedii 4A strain is capable of synthesizing EPS that was confirmed by the Podobedov-Molish reaction (a qualitative reaction to carbohydrates in the medium) on a medium supplemented with 5% sucrose. Gel permeation chromatography confirmed that the isolated strain produces an exopolysaccharide with a molecular weight of about 1.69 MDa on a medium supplemented with 5% sucrose. However, the presence of two lower molecular weight peaks may indicate that the resulting product has heterogeneous structure. The yield of dry biopolymer when growing the strain on a mineral medium with sucrose at a final concentration of 5% without adding additional components, such as microelements, was 8.5 g/l. One of the important characteristics of EPS is the viscosity of their aqueous solutions. The measured dynamic viscosity of a 10% EPS solution synthesized by Pantoea cypripedii 4A was 1.728 mPa s. Despite the fact that the production of extracellular polysaccharides is often enhanced under stress conditions, neither reduced oxygen concentration, nor low temperature conditions did affect the EPS biosynthesis by the studied strain.

1. Гвоздяк, Р.И. (1989). Микробный полисахарид ксантан. Наукова думка.

2. Елинов, Н.П. (1984) Химия микробных полисахаридов. Высшая школа.

3. Мелентьев, А.И. (2017). Уникальный природный штамм Paenibacillus ehimensis IB-739. Нарочанские чтения - 11. (с. 58-63). Белорусский государственный университет, Северо-Кавказский федеральный университет.

4. Пирог, Т.П., Гринберг, Т.А., & Малашенко, Ю.Р. (1997). Защитные функции экзополисахаридов, синтезируемых бактериями Acinetobacter sp. Микробиология, 66(3), 335 – 340.

5. Середа, А.С., Костылева, Е.В., Великорецкая, И.А., Цурикова, Н.В., Хабибулина, Н.В., Бикбов, Т.М., Бикбов, Т.М., Бубнова, Т.В. & Немашкалов, В.А. (2019). Использование препарата на основе низкомолекулярных веществ сои для повышения активности ксиланазы и эндоглюканазы мутантного штамма Trichoderma reesei Co-44. Биотехнология, 35(5), 70 – 79. https://doi.org/10.21519/0234-2758-2019-35-5-70-79

6. Andhare, P., Chauhan, K., Dave M., & Pathak, H. (2014). Microbial exopolysaccharides: advances in applications and future prospects. Biotechnology, 3, 1–25. https://doi.org/10.13140/RG.2.1.3518.4484

7. Amellal, N., Burtin, G., Bartoli, F., & Heulin, T. (1998). Colonization of wheat roots by an exopolysaccharide-producing Pantoea agglomerans strain and its effect on rhizosphere soil aggregation Appllied Environmental of Microbiolology, 64, 3740–3747.

8. Banerjee, A., Rudra, S.G., Mazumder, K., Nigam, V., & Bandopadhyay, R. (2018). Structural and functional properties of exopolysaccharide excreted by a novel Bacillus anthracis (Strain PFAB2) of hot spring. Original Indian Journal of Microbiology, 58, 39–50, https://doi.org/10.1007/s12088-017-0699-4

9. Barcelos, M. C. S., Vespermann, K. A. C., Pelissari F. M., & Molina G. (2020). Current status of biotechnological production and applications of microbial exopolysaccharides. Critical Reviews in Food Science and Nutrition, 60(9), 1475-1495, https://doi.org/10.1080/10408398.2019.1575791

10. Benit, N., & Roslin, A.S. (2018). Isolation and characterization of larvicidal extracellular polysaccharide (EPS) from Pseudomonas aeruginosa B01. International Journal of Current Microbiology and Applied Sciences, 7, 109-120, https://doi.org/10.20546/ijcmas.2018.701.013

11. Birch, J., Van Calsteren, M.-R., Pérez, S. & Svensson, B. (2019). The exopolysaccharide properties and structures database: EPS-DB. Application to bacterial exopolysaccharides. Carbohydrate Polymers, 205, 565–570. https://doi.org/10.1016/j.carbpol.2018.10.063

12. De Bruyne, K., Slabbinck, B., Waegeman, W., Vauterin, P., De Baets, B., & Vandamme, P. (2011). Bacterial species identification from MALDI-TOF mass spectra through data analysis and machine learning. Systematic and Applied Microbiology, 34, 20 – 29. https://doi.org/10.1016/j.syapm.2010.11.003

13. Carrion, O., Delgado, L., & Mercade, E. (2015). New emulsifying and cryoprotective exopolysaccharide from Antarctic Pseudomonas sp. ID1. Carbohydrate polymers, 117, 1028 – 1034. https://doi.org/10.1016/j.carbpol.2014.08.060

14. Castellane, T. C., Lemos, M., & Lemos, E. (2018). Exploring and utilization of some bacterial exopolysaccharides. Biopolymers Research, 2(1), 1000106

15. Chrismas, N., Barker, A. G., & Anesio, A.M. (2016). Genomic mechanisms for cold tolerance and production of exopolysaccharides in the Arctic cyanobacterium Phormidesmis priestleyi BC1401. BMC Genomics, 17(1), 533. https://doi.org/10.1186/s12864-016-2846-4

16. Guezennec, J. (2016). Bacterial exopolysaccharides from unusual environments and their applications. In H.C. Flemming, T.R. Neu, J. Wingender (Eds) The Perfect Slime: Microbial Extracellular Polymeric Substances (EPS) (p.135). N. Y.: Springer.

17. Leroy, F., & De Vuyst, L. (2016). Advances in production and simplified methods for recovery and quantification of exopolysaccharides for applications in food and health. Journal of Dairy Science, 99(4), 3229 – 3238. https://doi.org/10.3168/jds.2015-9936

18. Leung, M.Y., Liu, C., Koon, J.C., & Fung, K.P. (2006). Polysaccharide biological response modifiers. Immunology Letter, 105(2), 101-114. https://doi.org/10.1016/j.imlet.2006.01.009.

19. Marx, J.G., Carpenter, S.D., & Deming, J.W. (2009). Production of cryoprotectant extracellular polysaccharide substances (EPS) by the marine psychrophilic bacterium Colwellia psychrerythraea strain 34H under extreme conditions. Canadian Journal of Microbiology, 55(1), 63 –72. https://doi.org/10.1139/W08-130

20. Matsumoto, Y., & Kuroyanaqi, Y. (2010). Development of a wound dressing composed of hyaluronic acid sponge containing arginine and epidermal growth factor. Journal of Biomaterials Science Polymer, 21, 715–726. https://doi.org/10.1163/092050611X555687

21. Niknezhad, S. V., Morowvat, M. H., Najafpour D., G., Iraji, A., & Ghasemi, Y. (2018). Exopolysaccharide from Pantoea sp. BCCS 001 GH isolated from nectarine fruit: production in submerged culture and preliminary physicochemical characterizations. Food Science and Biotechnology, 27, 1735–1746. https://doi.org/10.1007/s10068-018-0409-y

22. Okonechnikov, K., Golosova, O., Fursov, M., & UGENE team (2012). Unipro UGENE: a unified bioinformatics toolkit. Bioinformatics (Oxford, England), 28(8), 1166–1167. https://doi.org/10.1093/bioinformatics/bts091

23. Ostapska, H., Howell, P.L., & Sheppard, D.C. (2018) Deacetylated microbial biofilm exopolysaccharides: It pays to be positive. PLOS Pathogens, 14(12), e1007411. https://doi.org/10.1371/journal.ppat.1007411

24. Roca, C., Alves, V.D., Freitas, F., & Reis, M.A. (2015). Exopolysaccharides enriched in rare sugars: bacterial sources, production, and applications. Frontiers in microbiology, 6, 288 – 291. https://doi.org/10.3389/fmicb.2015.00288

25. Sambrook, J., Fritsch, E.F., & Maniatis, T. (1989). Molecular cloning: a laboratory manual. 2nd ed. N.Y.: Cold Spring Harbor Laboratory Press.

26. Silva, L.A., Lopes Neto, J.H.P. & Cardarelli, H.R. (2019). Exopolysaccharides produced by Lactobacillus plantarum: technological properties, biological activity, and potential application in the food industry. Annual Microbiology, 69, 321–328 https://doi.org/10.1007/s13213-019-01456-9

27. Silvi, S., Barghini, P., Aquilanti, A., Juarez-Jimenez, B., & Fenice, M. (2013). Physiologic and metabolic characterization of a new marine isolate (BM39) of Pantoea sp. producing high levels of exopolysaccharide. Microbial Cell Factories, 12(1), 10. https://doi.org/10.1186/1475-2859-12-10

28. Sun, L., Lei, P., Wang, Q., Ma, J., Zhan, Y., Jiang, K., Xu, Z. & Xu, H. (2020). The endophyte Pantoea alhagi NX-11 alleviates salt stress damage to rice seedlings by secreting exopolysaccharides. Frontiers in Microbiology, 10, 3112. https://doi.org/10.3389/fmicb.2019.03112

29. Sutherland, I.W. (2005). Microbial exopolysaccharides. In Polysaccharides: structural diversity and functional versatility.(pp. 431-457). N.Y.: Marcel Dekker, Inc.

30. Trabelsi, I., Slima, S.B., Chaabane, H., & Riadh, B.S. (2015). Purification and characterization of a novel exopolysaccharides produced by Lactobacillus sp. Ca6. International Journal of Biological Macromolecules, 74, 541–546. https://doi.org/10.1016/j.ijbiomac.2014.12.045

31. De Vuyst, L., & Degeest, B. (1999). Heteropolysaccharides from lactic acid bacteria. FEMS Microbiology Reviews. 23, 153–177. https://doi.org/10.1016/S0168-6445(98)00042-4

32. Weisburg, W.G., Barnes, S.M., Pelletier, D.A., & Lane, D.J. (1991). 16S ribosomal DNA amplification for phylogenetic study. Journal of Bacteriology, 73, 697–703.